Comparative metabolic profiling of olive leaf extracts from twelve different cultivars collected in both fruiting and flowering seasons

Olea europaea is an economically significant crop native to Mediterranean countries. Its leaves exhibit several biological properties associated to their chemical composition. The aqueous ethanolic extracts of olive leaves from twelve different cultivars were analyzed by high performance liquid chromatography coupled to photodiode array and electrospray ionization mass spectrometry (HPLC/PDA/ESI–MS/MS). A total of 49 phytochemicals were identified in both positive and negative ionization modes. The identified compounds belonged to four classes of secondary metabolites including secoiridoids, flavonoids, pentacyclic triterpenoids and various phenolic compounds. Seasonal variation in chemical composition among the studied cultivars was apparent in autumn and spring. Secologanoside, oleuropein, hydroxy-oleuropein, demethyl oleuropein, gallocatechin, luteolin-O-hexoside, diosmetin, oleanolic acid and maslinic acid were detected in all cultivars in both seasons. Oleuropein-O-deoxyhexoside was tentatively identified for the first time in olive leaf extracts; detected only in the Spanish cultivar Picual (PIC) collected in spring. Also, dihydroxy-oxooleanenoic acid and hydroxy-oxooleanenoic acid, two bioactive pentacyclic triterpenes, were identified. Principle component analysis (PCA) showed good discrimination among the studied cultivars in terms of their botanical origin. This study is considered the first study for non-targeted metabolic profiling of different olive leaf cultivars cultivated in Egypt.


Results and discussion
HPLC-PDA-ESI-MS/MS analysis. The chemical composition of olive leaf extracts from twelve cultivars collected in autumn and spring were analyzed using HPLC coupled to ion trap mass spectrometer with an ESI source. All extracts were analyzed in both positive and negative modes to cover compounds with diverse ionization responses. ESI − has been previously used to determine the structure of flavonoid glycosides 24,25 , iridoids, and triterpenes; whereas, coumarins, and alkaloids showed better ionization in ESI + positive mode 26,27 . A comprehensive metabolite profiling was performed for all olive leaf extracts. A total of 49 metabolites were annotated belonging to 4 different classes including secoiridoids, flavonoids, triterpenoids, and various other phenolic compounds. Table 1 shows the compounds that were tentatively identified in O. europaea leaf extracts. The elution order is based mainly on their polarity, the more polar the compound the shorter the retention time. The total ion chromatogram of the twelve cultivars collected in autumn (A) and spring (B), in the negative ionization mode is presented in Fig. 1. The base peak chromatograms of individual olive leaf extracts analyzed in negative ionization mode in both seasons are displayed in supplementary figure (Fig. S1). Structures of selected metabolites identified in O. europaea leave extracts belonging to secoiridoids (A), flavonoids (B) and pentacyclic triterpenes (C) were illustrated in Fig. 2.
Secoiridoids. Oleaeuropaea is rich in secoiridoids; especially the esterified forms with a phenolic moiety are known as oleosides 28 . Lack of characteristic chromophores for most of secoiridoids renders UV spectra usually of limited use 29 . However, secoiridoids are characterized by marked absorption bands at λ max 240 nm and 270 nm 30 . HPLC-MS fragmentation pathways were used to determine the molecular formula and the loss of characteristic moieties. The fragment ions appeared are corresponding to distinctive losses, such as [M−H− CH 3  Oleoside derivatives. Compounds (4) and (5) eluted at R t 10.04 and 14.69 min, respectively, showed a molecular ion peak [M−H] − at m/z 389 related to oleoside and its isomer secologanoside. Their MS 2 spectra (Supp. Figs. S2, S3) exhibited a fragment ion peak at m/z 345 arising from the loss of CO 2 (44 Da) of a carboxylic group while the product ion at m/z 277 was related to the loss of a hexose moiety (162 Da). A fragment ion peak at m/z 183 indicated a subsequent loss of CO 2 . This fragmentation pattern emphasizes the presence of two carboxylic groups and a hexose moiety. Secologanoside is eluted after oleoside in reversed phase conditions and exhibits a strong peak at m/z 345 32 33 . Elenolic acid is considered as a degradation product of oleuropein and is used as a marker for olive maturation 34 . It is worth mentioning that elenolic acid could not be detected in all leaf extracts during spring.
Oleuropein derivatives. Nine known oleuropein derivatives were identified, exhibiting similar UV absorption maxima and mass fragmentation pattern 35 36 . It has a structure similar to oleuropein with additional ethoxide moiety (m/z 583.09). MS 2 spectrum showed a fragment ion peak at m/z 537 relative to the loss of an ethanol moiety [M−H-46] − , followed by similar fragmentation pattern to oleuropein (m/z 375, 305, 273).
Compound (37) was identified as ligstroside; a deoxy analogue of oleuropein; with a molecular ion peak at m/z 523.17 and fragment ion peaks at (m/z 361, 291, 259) 17 . Compound (31)  Flavonoids. Various flavonoids were previously reported in olive leaf extract, either in aglycone or glycosylated forms. In this work flavonoid identifications were based on studying their UV spectra and the mass spectrum of each identified compound.
Flavones. The UV/Vis spectra of flavones characterized by a λ max for band Ι around 340 nm, as well it provides valuable information about the degree of hydroxylation as the increase in the number of hydroxyl groups increased λ max 38 . Their MS spectra used to determine molecular formula and identify the structure of the aglycone for the eluted flavonoid based on its fragmentation pattern.
Compound (39) (18) was tentatively identified as luteolin-O-robinoside eluting before its isomer luteolin-O-rutinoside (19); that was previously isolated from olive leaf extracts 39 . A peak at R t 38.62 min was obvious in all the examined cultivars. It showed a UV absorption maximum at 345 nm for band I characteristic for flavones. The EIC at m/z 447.11 with its strong fragment at m/z 285, due to the loss of a hexose residue, indicates luteolin-O-hexoside (22). Luteolin-7-O-glucoside was previously detected as a major compound in olive leaf extracts as well as in the fruits 17 . In parallel, the MS 2 spectra of compounds 25, 26, 29 and 30 exhibited a fragment ion peak at m/z 269 corresponding to apigenin moiety. Compound (25)  Additional phenolics and phenolic acids. Compound (3) showed a UV spectrum with two λ max at 220 and 283 nm. It exhibited a molecular ion peak [M-H] − at m/z 153 and a fragment ion peak at m/z123 corresponding to the loss of a CH 2 OH group. This was consistent with hydroxytyrosol, one of the main components of olive leaves, as previously described 41 (44 Da), followed by the loss of CO (28 Da) and another molecule of CO 2 , thus, this compound was tentatively identified as ellagic acid 42 . Compound (27) showed a molecular ion peak [M−H] − at m/z 623.25 with its product ions at m/z 461 due to loss of caffeic acid moiety, weak ion at m/z 315 due to loss of rhamnose unit and a fragment ion at m/z 161 due to proton transfer of the remaining ketene fragment. This is coincident with that reported for verbascoside fragmentation pattern 33 . Thus, compound 27 was identified as verbascoside;a heterosidic ester of caffeic acid and hydroxytyrosol which was previously detected in appreciable amount in mature olive leaves 44 .
Pentacyclic triterpenoids. O. europaea fruit and leaf have been reported as a rich source of triterpenic acids and pentacyclic triterpenols either free or esterified with fatty acids 45 . Among these, oleanolic, ursolic, maslinic acids are the most prominent triterpene acids in the olive leaves, as well as, uvaol, erythrodiol as triterpene alcohols 46  Although most interests are directed toward the phenolic composition of olive leaf extract; olive leaves have been reported as a rich source of bioactive pentacyclic triterpenes 45 . Triterpenes are characterized by diverse pharmacological properties including hepatoprotective, anti-inflammatory, antimicrobial, anti-hyperlipidemic, gastro protective and antidiabetic effects 47 . Guinda et al. 2010b determined the triterpenoid content of the fruits and leaves of three Spanish cultivars. Results showed that the levels of triterpenes in the leaf extract were 30 fold higher than those found in the fruit 45 .
Herein, the triterpenoid contents studied for different cultivars were shown to be dependent on the variety, and in all cases oleanolic acid was the major triterpenic compound. Hydroxy-oxooleanenoic acid (45) and dihydroxy-oxooleanenoic acid (44), are first identified in olive leaf extracts according to our knowledge. They were identified in all the studied cultivars. Hydroxy-oxooleanenoic acid and its derivatives were shown to possess antimicrobial and cytotoxic activities against wide tumor cell lines 48 . Thus, olive leaf extract might serve as a source for such valuable anti-tumor agent.
Multivariate data analysis of HPLC-MS data. PCA as unsupervised multivariate data analysis technique was performed to explain metabolite differences and possible discrimination between the studied cultivars in an untargeted manner. The aligned peak lists obtained from the processing of HPLC-MS data of the negative ionization mode for autumn and spring extracts were subjected to PCA analysis. The score plot obtained for autumn extracts (Fig. 3A) showed two orthogonal PCs, accounting for 47% of the variance among the data. The score plot showed marked segregation among cultivars in relation to their botanical origin, where the three Spanish cultivars located positive to PC 2 , while the Egyptian cultivars are clustered in the negative side. The two Greek cultivars (KAL and KOR) are grouped together in the upper left quadrant. The PCA was able to differentiate between Egyptian cultivars and others that reveal differences in their composition based on their botanical origin. By examining the loading plot (Fig. 3B) to explain the underlying reasons for such clustering; flavonoids and secoiridoids were found to contribute the most in species discrimination. They segregate the cultivars into two groups one positive to PC 1 for cultivars rich in secoiridoids including MAN, PIC, AOK and ASH. Another group clustered negative to PC 2 includes (ABQ, KOR, KAL, COR, TFH, MRK, HMD and WAT) related to their high flavonoids. Concerning to the identified metabolites, diosmetin was found to be more enriched in KOR, KAL, ABQ and TFH. On the other hand, the two Spanish cultivars MAN and PIC were found to be rich in secologanoside, oleoside and oleuropein-O-hexoside; whereas oleuropein, oleuropein aglycone and luteolin-O-hexoside were found to be more enriched in the Egyptian cultivars ASH and AOK. www.nature.com/scientificreports/ PCA analysis for spring extracts was performed. The obtained score and loading plots (Fig. 4A,B), showed the segregation of cultivars based on their metabolites. Here again flavonoids and secoiridoids were found to contribute the most in species discrimination. Negative ion-mode MS, in which metabolites are deprotonated has the potential to increase the coverage of phenolic compounds analysis 49 . In the present study, most of phenolics are more likely to retain a negative charge and thus large amount of data generated in the negative mode. Therefore, negative ionization data has been chosen for principal component analysis (PCA). Two groups were observed; one positive to PC 2 includes MAN, COR, KAL, KOR, HMD, ASH and MRK. The second group clustered negative to PC 2 includes AOK, ABQ, PIC, WAT and TFH. In terms of the identified metabolites oleuropein and oleuropein-O-hexoside were found to be more abundant in the first group, while the second group was found to be richer in apigenin-O-hexoside and luteolin.

Seasonal variation in olive leaf composition. By examining the TIC for all extracts in both seasons
in the phenolic region, it was obvious that spring is characterized by higher oleuropein content for most of the studied cultivars. This can be correlated with the absence of elenolic acid; a degradation product of oleuropein; in all leaf extracts during spring. Certain compounds were identified in all the studied olive cultivars leaf extract. They were present in all cultivars in both seasons but differ quantitatively. They include secologanoside (5), gallocatechin (7), elenolic acid hexoside (9), hydroxy-oleuropein (12), demethyl oleuropein (13) (44), hydroxy-oxooleanenoic acid (45), oleanolic acid (46), and maslinic acid (49). Oleuropeinic acid (31) was present in specific cultivars in autumn (MAR, WAT, MAN, PIC, KOR) and was not detected in spring except for PIC. Nuezhenide (14) was known as the major phenolic compound in olive seeds 50 . It was detected previously in the leaves of the unique Australian olive cultivar Hardy's Mammoth and certain Spanish cultivars 39 . Herein, nuezhenide was detected only in the leaf extracts of ASH and PIC in both seasons in addition to the two Greek cultivars (KOR, KAL) during spring.
To the best of our knowledge, a new compound namely, oleuropein-O-deoxyhexoside (23), was tentatively identified for the first time in nature. Oleuropein-O-deoxyhexoside (23); was detected only in the Spanish cultivar PIC during spring with marked decrease in oleuropein peak. Moreover, ethyl gallate (6) was detected only in ABQ during spring and its activity in the protection against diabetes has been previously reported 43 . In contrast to possible expectations, oleuropein was not the major compound detected in all the studied cultivars. Luteolin-O-hexoside was shown to be more predominant in some extracts. It was the main compound in HMD, WAT, ABQ, KOR during autumn; and TFH, PIC during spring.  For this study, a sample of each cultivar was collected from three different trees, (n = 3) was obtained. After air drying, the leaves were grounded in a rotor mill and the dried powders were stored at 4 °C protected from light and moisture. The code, name and origin of each cultivar are shown in Table 2.
Chemicals and reagents. Absolute ethanol of HPLC grade was obtained from fisher scientific, UK; Acetonitrile, methanol and formic acid (LC-MS grade) were obtained were obtained from Sigma-Aldrich, Steinheim, Germany, milliQ water was used for HPLC analysis. All other chemicals were purchased from Sigma-Aldrich (Merck, USA).

Extracts preparation for HPLC-MS analysis.
Selection of the most appropriate extraction method was based on previous work, where the highest yield of phenolic compounds was obtained using aqueous alcoholic solution [51][52][53] . The dried powders obtained for each cultivar were percolated in 70% ethanol for one day then filtered and the process was repeated two times in three consecutive days. The obtained liquid extract for each cultivar was concentrated with rotary evaporator (Büchi, Switzerland) and completely dried using a lyophilizer (Christ, Alpha 1-2 LD Plus) to yield a dry powder for each extract. The obtained powders were re-suspended in methanol for LC/MS analysis. www.nature.com/scientificreports/ from 2 to 100% acetonitrile in 60 min at 30 °C. The flow rate was 0.5 ml/min. The injection volume was about 20 µl. All samples were measured in the positive and negative mode. The MS was operated with a capillary voltage of 10 V, source temperature of 240 °C, and high purity nitrogen as a sheath and auxiliary gas at a flow rate of 80 and 40, respectively. The ions were detected in a mass range of 50-2000 m/z. Collision energy of 35% was used in MS/MS for fragmentation. Data acquisitions and analyses were executed by Xcalibur™ 2.0.7 software (Thermo Scientific). Olive leaf extracts were analyzed in both positive and negative ionization modes. Metabolites identification was based on comparing the retention time, UV/Vis and mass spectra of each eluted compound with those reported in literature and online databases 54-57 . HPLC-ESI-MS data processing and multivariate data analysis. The whole mass profile for each cultivar was processed using MZmine2 version 2. 39, an open-source software that is used for visualization and analysis of mass spectrometry based molecular profile data 58 . Thermo raw files obtained from Xcalibur are converted to NetCDF and then imported to MZmine software. LC/MS data processing based on chromatogram building and deconvlusion to individual peaks then aligned using RANSAC aligner. The resultant aligned peak list was further processed for gap filling step then exported to Microsoft Excel software to construct a data matrix containing all the aligned peaks m/z with their retention time and peak areas. The excel data matrix was subjected to PCA using the unscramble software to detect possible discrimination between cultivars and also to determine the main components responsible for the discrimination. All variables were mean centered and scaled to Pareto variance.

Data availability
Data are available upon request from the first author, Eman M. Kabbash. www.nature.com/scientificreports/